There is an urgent need for the development of leukaemia-targeted immunotherapeutic approaches. Wilms’ tumour antigen (WT1), M-phase phosphoprotein 11 (MPP11) and proteinase-3 (PR-3) proteins are overexpressed in leukemic cells and represent attractive immunotherapeutic candidates. The first part of this study explored the feasibility of using an approach to develop a novel leukaemia vaccine by modifying the sequence of low avidity HLA-A*0201- restricted peptide epitopes derived from WT1 protein (WT1-126126-134 and WT1- 187187-195). The modified WT1-Db126 showed enhanced binding ability to the HLA-A*0201 molecule, increased the frequency of IFN-γ producing cytotoxic T lymphocyte (CTL), and boosted the lytic activity of the generated CTL against HLA-matched leukaemia cells. Interestingly, the CTL line generated with the modified epitope was able to recognize the wild-type peptide presented by target cells. The second part of this study identified a novel epitope derived from WT1 antigen (WT1-60237-251). This epitope was recognized by a CD4+ T cells in an HLA-DRB1*04-restricted manner and secreted Th2 cytokines (IL-5 and IL-4). The third part of the study aimed to identify potential CD8+ CTL epitopes in the sequence of MPP11 and PR-3 that may bind to HLA-A*0201 molecule and provoke specific CTL responses. A potential HLA-A*0201 binding epitope named MPP-437- 45 derived from the MPP11 protein was identified which was used to generate a CTL line. This CTL line specifically recognized peptide-loaded target cells in both ELISPOT and cytotoxic assays. Importantly, this CTL line exerted a cytotoxic effect towards the CML leukemic cell line K562-A2.1. The study also demonstrated that PR-3-derived peptides PR-129129-137 and PR-99-17 were not immunogenic since they were incapable of inducing specific CD8+ T cell responses. In conclusion, modification of the WT1-Db126 epitope resulted in enhancement of its immunogenicity without altering its antigenic specificity. The WT1-60 and MPP- 4 peptides have been identified as novel CD4 and CD8 T-cell epitopes, respectively.